STK25 is a candidate gene for pseudopseudohypoparathyroidism.

We determined the chromosomal location of the mouse gene Stk25, encoding a member of the Ste20/PAK family of serine/threonine kinases, by interspecific backcross analysis. We mapped Stk25 to the central region of mouse chromosome 1 linked to Chrng (formerly Acrg) and En1. This central region of mouse chromosome 1 shares a region of homology with the long arm of human chromosome 2, suggesting that the human homologue of Stk25 would also map to 2q. We proved this prediction of syntenic homology correct by mapping human STK25 to 2q37. Deletion of the 2q37 region has been implicated in the expression of pseudopseudohypoparathyroidism (PPHP), a disease which shares features of the Albright hereditary osteodystrophy (AHO) phenotype. To investigate a pathogenetic relationship between STK25 and PPHP, we carried out fluorescence in situ hybridization (FISH) using an STK25 gene probe and chromosomes from PPHP patients characterized as having small deletions near the distal end of 2q. PPHP patient DNA showed no hybridization to STK25 genomic DNA, indicating that STK25 is contained within the deleted chromosomal region. This finding, in conjunction with previous studies demonstrating the role of Ste20/PAK kinases in heterotrimeric G protein signaling, suggests that STK25 is a positional candidate gene for PPHP.

Nuclear import of activated D-ERK by DIM-7, an importin family member encoded by the gene moleskin.

The initiation of gene expression in response to Drosophila receptor tyrosine kinase signaling requires the nuclear import of the MAP kinase, D-ERK. However, the molecular details of D-ERK translocation are largely unknown. In this regard, we have identified D-Importin-7 (DIM-7), the Drosophila homolog of vertebrate importin 7, and its gene moleskin. DIM-7 exhibits a dynamic nuclear localization pattern that overlaps the spatial and temporal profile of nuclear, activated D-ERK. Co-immunoprecipitation experiments show that DIM-7 associates with phosphorylated D-ERK in Drosophila S2 cells. Furthermore, moleskin mutations enhance hypomorphic and suppress hypermorphic D-ERK mutant phenotypes. Deletion or mutation of moleskin dramatically reduces the nuclear localization of activated D-ERK. Directly linking DIM-7 to its nuclear import, this defect can be rescued by the expression of wild-type DIM-7. Mutations in the Drosophila Importin beta homolog Ketel, also reduce the nuclear localization of activated D-ERK. Together, these data indicate that DIM-7 and Ketel are components of the nuclear import machinery for activated D-ERK.

KDR activation in astrocytic neoplasms.

BACKGROUND: The development of new capillary networks appears to be necessary for the growth of solid tumors. Tumor angiogenesis is believed to be mediated by soluble factors released from tumor cells that then act on endothelial cells in a paracrine manner. Vascular endothelial growth factor (VEGF) is a prime regulator of normal and tumor angiogenesis as well as vasculogenesis. VEGF is expressed in glioma cells and its receptors (Flt-1 and KDR) are expressed in the same gliomas. The two receptors are tyrosine kinases and have an extracellular domain containing seven immunoglobulin-like loops and a split tyrosine-kinase domain. KDR is a receptor for the various VEGF isoforms and for VEGF-C; Flt-1 is a receptor for the various isoforms. Studies suggest that the VEGF receptors are induced in endothelial cells during tumor angiogenesis. Stimulation of aortic endothelial cells results in receptor tyrosine phosphorylation (receptor activation). In this study the activation state of the KDR receptors was determined in low grade, anaplastic, and high grade gliomas. METHODS: A synthetic tyrosine phosphopeptide was used to raise an antibody that recognizes the phosphorylation state of tyrosine 1054/1059 in the KDR receptor. Western blot analysis was performed on 37 astrocytic neoplasms (7 low grade astrocytomas, 13 anaplastic astrocytomas, and 17 cases of glioblastoma multiforme). RESULTS: Immunoblotting with this antibody found that tyrosines 1054/1059 were phosphorylated constitutively within multiple fresh surgical specimens of glioblastomas (71%) and anaplastic gliomas (15%), but not in low grade gliomas. CONCLUSIONS: The findings of the current study strongly support the hypothesis that the onset of angiogenesis is an important event during the disease progression of gliomas.

A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle.

In Drosophila melanogaster, position-effect variegation of the white gene has been a useful phenomenon by which to study chromosome structure and the genes that modify it. We have identified a new enhancer of variegation locus, Dmrnahel (hel). Deletion of mutation of hel enhances white variegation, and this can be reversed by a transformed copy of hel+. In the presence of two endogenous copies, the transformed hel+ behaves as a suppressor of variegation. hel is an essential gene and functions both maternally and zygotically. The HEL protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential HEL homologues have been found in mammals, yeast and worms. HEL protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. We propose that the HEL protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that HEL is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis.

The nonreceptor protein tyrosine phosphatase corkscrew functions in multiple receptor tyrosine kinase pathways in Drosophila.

Corkscrew (csw) encodes a nonreceptor protein tyrosine phosphatase (PTPase) that has been implicated in signaling from the Torso receptor tyrosine kinase (RTK). csw mutations, unlike tor mutations, are associated with zygotic lethality, indicating that Csw plays additional roles during development. We have conducted a detailed phenotypic analysis of csw mutations to identify these additional functions of Csw. Our results indicate that Csw operates positively downstream of other Drosophila RTKs such as the Drosophila epidermal growth factor receptor (DER), the fibroblast growth factor receptor (Breathless), and likely other RTKs. This model is substantiated by specific dosage interactions between csw and DER. It is proposed that Csw is part of the evolutionarily conserved "signaling cassette" that operates downstream of all RTKs. In support of this hypothesis, we demonstrate that SHP-2, a vertebrate PTPase similar to Csw and previously implicated in RTK signaling, encodes the functional vertebrate homologue of Csw.

Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs.

We have identified a putative Drosophila STAT protein named Marelle that exhibits mutant phenotypes identical to mutations in the Hopscotch/JAK kinase. We show that a reduction in the amount of marelle gene activity suppresses the phenotype associated with a gain-of-function mutation in hopscotch and enhances the phenotype associated with a weak hopscotch mutation. We propose that Hopscotch activates Marelle to regulate transcription of target genes such as the pair rule gene even-skipped. Our results demonstrate the existence of an invertebrate JAK/STAT system.

Dissection of the Torso signal transduction pathway in Drosophila.

Cell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP-binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D-Raf and D-Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane-bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D-Raf and D-Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D-Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D-raf mutation that abolishes binding of Ras1 to D-Raf. These findings indicate that full D-Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1-independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso.

The torso receptor tyrosine kinase can activate Raf in a Ras-independent pathway.

Activation of the receptor tyrosine kinase (RTK) torso defines the spatial domains of expression of the transcription factors tailless and huckebein. Previous analyses have demonstrated that Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. By using a recently developed technique of germline mosaics, we find that D-Raf can be activated by torso in the complete absence of Ras1. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) or the adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Ras1 to D-Raf. Our study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway.

Genetic and molecular analyses of mutations involved in Drosophila raf signal transduction.

We have identified dominant mutations that suppress the lethality associated with an R217-->L mutation in the GTP.Ras binding region (CR1) of the Drosophila raf (D-raf) serine/threonine kinase. Four intragenic and seven extragenic suppressors were recovered. Each of the four intragenic mutations contains one compensatory amino acid change located in either the CR1 or the kinase domain of D-raf. The seven extragenic suppressors represent at least four genetic loci whose effects strongly suggest that they participate in both the sevenless and Drosophila EGF receptor (DER) signaling pathways. One of these mutations, Su(D-raf)34B, is an allele of D-mek which encodes the known signaling molecule MAPK kinase (MEK). A D83V mutation in D-MEK is identified and shown to be sufficient to confer the dominant activity of Su(D-raf)34B.

The Drosophila stubarista phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless.

We describe the molecular characterization of the Drosophila melanogaster gene stubarista (sta) that encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. In addition, we have analyzed the basis of the sta1 visible phenotype which consists of shortened antennae and bristles. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. We provide genetic evidence that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. We demonstrate that sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein.

Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase.

Formation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase corkscrew, controls the domains of expression of the transcription factors tailless and huckebein. In this paper, we report the molecular and developmental characterization of mutations in the D-raf gene. We show that mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that D-raf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wild-type amount of D-raf protein.

Structure and sequence of the calmodulin gene from Neurospora crassa.

cDNA and genomic clones of Neurospora calmodulin were obtained by PCR. Characterization revealed an open reading frame encoding a predicted protein of 149 amino acids, showing 85% identity to the human calmodulin protein sequence. Comparison of the cDNA and genomic sequence reveals the position of five introns, organized differently than is found in calmodulin genes from higher eukaryotes.

The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins.

The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.

Studies on herpes simplex virus and cancer.

Virus-induced polypeptides of cells infected by herpes simplex virus (HSV) types 1 and 2 were investigated by analysis on polyacrylamide gels and by determination of their antigenicity. Some polypeptides, VP154 and VP134, had immunological reactivity common to both virus types, while others (VP175 and VP123) were type specific. Only the glycosylated polypeptides were able to induce neutralizing antibody. The expression of viral genetic information was studied in newborn mice infected with wild-type and ts mutant viruses; some mutants had become attenuated and had lost pathogenicity for newborn mice while others had not. From induction experiments in HSV=transformed hamster cells, it appears that detection of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic information. Sera obtained from cancer patients were examined for antibodies to early proteins synthesized in HSV-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. Cervical cancer patients had sera with a higher reactivity to early nonstructural polypeptides than to breast cancer patients or to matched healthy women. In contrast to the results with early polypeptides, little difference was detectable between the matched sera in their reactivity with a major capsid polypeptide, which is synthesized late in the infectious cycle.

Biophysical studies of vesicular stomatitis virus.

McCombs, Robert M. (Baylor University College of Medicine, Houston, Tex.), Matilda Benyesh-Melnick, and Jean P. Brunschwig. Biophysical studies of vesicular stomatitis virus: J. Bacteriol. 91:803-812. 1966.-The infectivity and morphology of vesicular stomatitis virus (VSV) were studied after density gradient centrifugation in cesium chloride (CsCI), potassium tartrate (KT), and sucrose. Centrifugation in CsCl revealed two equally infectious bands corresponding to densities of 1.19 and 1.22 g/ml, and a third (density, 1.26 g/ml) band of low infectivity. Two bands (densities of 1.16 and 1.18 g/ml) were observed in the KT gradient, in which the lighter band contained most of the infectivity. Centrifugation in sucrose resulted in a single broad infectious band (density, 1.16 g/ml). The typical rod-shaped VSV particles were found mainly in the lighter bands obtained in CsCl (1.19 g/ml) and KT (1.16 g/ml) and in the single sucrose gradient band (1.16 g/ml). Bent particles equally as infectious as the rod-shaped particles were a constant finding in the CsCl preparations, and were observed mainly in the second band (density, 1.19 g). Numerous strands 15mmu wide were found in the third CsCl (density, 1.26 g/ml) and the second KT (1.18 g/ml) bands. Similar strands could be liberated from VSV particles after treatment with deoxycholate. Internal transverse striations were found to be a regular feature of VSV particles examined with the pseudoreplication negative-staining technique. For crude virus stocks, the physical particle-to-infectivity ratio ranged from 73 to 194. Several morphological similarities between VSV and myxoviruses were observed, including 10 mmu surface projections, pleomorphic morphological forms, and 15 mmu seemingly nucleoprotein strands.